Processes for packaging oligonucleotides into virus-like particles of RNA bacteriophages

Abstract

The invention provides processes for the producing compositions comprising (i) a virus-like particle, wherein said virus-like particle is a virus-like particle of an RNA bacteriophage, and (ii) an oligonucleotide, wherein said oligonucleotide is packaged into said virus-like particle. The invention further provides processes for producing nucleotide compositions comprising oligonucleotides suitable to be used in the processes mentioned before. The invention further provides nucleotide compositions obtainable by the processes of the invention and uses thereof. The invention further provides compositions comprising (i) a virus-like particle, wherein said virus-like particle is a virus-like particle of an RNA bacteriophage, and (ii) an oligonucleotide, wherein said oligonucleotide is packaged into said virus-like particle, wherein said compositions are obtainable by the processes of the invention and wherein said compositions preferably comprises a purity of at least 98%, most preferably of at least 99%.

The invention claimed is: 
     
       1. A process for producing a composition comprising (i) a virus-like particle, wherein said virus-like particle is a virus-like particle of an RNA bacteriophage, and (ii) aggregated oligonucleotides, wherein said aggregated oligonucleotides are packaged into said virus-like particle, said process comprising the steps of:
 (a) generating a mixture, wherein said mixture comprises:
 (i) a coat protein of said RNA bacteriophage; 
 (ii) an agent capable of preventing the self-assembly of said coat protein; and 
 (iii) aggregated oligonucleotides, wherein said aggregated oligonucleotides comprise oligonucleotides comprising at least one poly G stretch, and wherein said aggregated oligonucleotides comprise a relative peak start time of 50 to 110%, where said relative peak start time is determined by size exclusion HPLC with the capsid of the RNA bacteriophage as the standard; 
 
 (b) removing said agent from said mixture; and 
 (c) allowing said coat protein to self-assemble into a virus-like particle. 
 
     
     
       2. The process of  claim 1 , wherein said coat protein comprises recombinant proteins, or fragments thereof that are capable of self-assembly, of a RNA bacteriophage. 
     
     
       3. The process of  claim 1 , wherein said RNA bacteriophage is Qβ. 
     
     
       4. The process of  claim 1 , wherein said RNA bacteriophage is AP205. 
     
     
       5. The process of  claim 1 , wherein said coat protein comprises a sequence selected from the group consisting of:
 (a) SEQ ID NO: 10 (Qβ CP); 
 (b) a mixture of SEQ ID NO:10 and SEQ ID NO:11 (Qβ A1 protein); 
 (c) SEQ ID NO: 12 (R17 coat protein); 
 (d) SEQ ID NO: 13 (fr coat protein); 
 (e) SEQ ID NO:14 (GA coat protein); 
 (f) SEQ ID NO: 15 (SP coat protein); 
 (g) a mixture of SEQ ID NO:15 and SEQ ID NO:16; 
 (h) SEQ ID NO: 17 (MS2 coat protein); 
 (i) SEQ ID NO: 18 (M11 coat protein); 
 (j) SEQ ID NO: 19 (MXI coat protein); 
 (k) SEQ ID NO:20 (NL95 coat protein); 
 (l) SEQ ID NO:21 (f2 coat protein); 
 (m) SEQ ID NO:22 (PP7 coat protein); and 
 (n) SEQ ID NO:23 (AP205 coat protein). 
 
     
     
       6. The process of  claim 1 , wherein the concentration of said oligonucleotides in said mixture is 25 to 100 μM. 
     
     
       7. The process of  claim 1 , wherein the molar ratio of said oligonucleotides and said coat protein in said mixture is 0.5 to 1.2. 
     
     
       8. The process of  claim 1 , wherein said agent comprises a denaturing compound selected from urea and guanidinium hydrochloride. 
     
     
       9. The process of  claim 1 , wherein said agent further comprises a reducing agent. 
     
     
       10. The process of  claim 1 , wherein said removing of said agent from said mixture is performed by a first buffer exchange with a first buffer, wherein said first buffer comprises sodium chloride. 
     
     
       11. The process of  claim 10 , wherein said first buffer exchange is performed across a membrane, wherein said membrane comprises a molecular weight cut off of 1 to 50 kD. 
     
     
       12. The process of  claim 1 , wherein said process further comprises the step of contacting said virus-like particle with an oxidizing agent, wherein said oxidizing agent is selected from the group consisting of:
 (a) hydrogen peroxide; 
 (b) oxygen; 
 (c) gluthathion; 
 (d) Cu 2+ ; and 
 (e) Fe 3+ . 
 
     
     
       13. The process of  claim 1 , wherein said process further comprises the step of purifying said virus-like particle, and wherein said purifying comprises a second buffer exchange with a second buffer, wherein said second buffer is a pharmaceutically acceptable buffer. 
     
     
       14. The process of  claim 13 , wherein said second buffer exchange is performed using a membrane, wherein said membrane comprises a molecular weight cut off of 100 to 1000 kD. 
     
     
       15. The process of  claim 1 , wherein the coat protein yield is at least 75% and/or wherein the oligonucleotide yield is at least 75%.